Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats

Endothelin-1 (ET-1), a potent vasoactive and mitogenic peptide, has been implicated in the pathogenesis of several forms of pulmonary hypertension. We hypothesized that nonspecific blockade of ET receptors would blunt the development of monocrotaline (MCT)-induced pulmonary hypertension in rats. A single dose of the nonspecific ET blocker bosentan (100 mg/kg) given to intact rats by gavage completely blocked the pulmonary vasoconstrictor actions of Big ET-1 and partially blunted hypoxic pulmonary vasoconstriction. After 3 wk, MCT-injected (105 mg/kg sc) rats gavaged once daily with bosentan (200 mg/kg) had lower right ventricular (RV) systolic pressure (RVSP), RV-to-body weight (RV/BW) and RV-to-left ventricular (LV) plus septal (S) weight [RV/(LV+S)] ratios and less percent medial thickness of small pulmonary arteries than control MCT-injected rats. Lower dose bosentan (100 mg/kg) had no effect on these parameters after MCT or saline injection. Bosentan raised plasma ET-1 levels but had no effect on lung ET-1 levels. Bosentan (200 mg/kg) also had no effect on wet-to-dry lung weight ratios 6 days after MCT injection. When given during the last 10 days, but not the first 11 days of a 3-wk period after MCT injection, bosentan reduced RV/(LV+S) compared with MCT-injected controls. We conclude that ET-1 contributes to the pathogenesis of MCT-induced pulmonary hypertension and acts mainly during the later inflammatory rather than the acute injury phase after injection.     

Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1

Administration of monocrotaline (MCT) causes pulmonary vascular lesions consisting of monocyte/macrophage infiltration in the early phase and medial thickening in pulmonary arteries and arterioles associated with pulmonary hypertension (PH) in the later phase. However, the molecular mechanism of monocyte/macrophage infiltration and its roles remain elusive. Herein, we have evaluated the role of a potent monocyte chemotactic and activating chemokine/monocyte chemoattractant protein-1 (MCAF/MCP-1) in MCT-induced PH in rats. A single injection of MCT induced PH at Day 21, as evidenced by increases in the ratio of right ventricular to left ventricular and septum weights (RV/LV+S) and right ventricular systolic pressure (RVSP). A significant increase in macrophage number in lungs started at Day 14, reaching a maximum at Day 21. MCAF/MCP-1 levels in bronchoalveolar lavage fluids were elevated significantly at Day 14 and remained high until Day 28, whereas plasma MCAF/MCP-1 levels increased at Day 7, returning to normal levels by Day 21. Immunoreactive MCAF/MCP-1 proteins were mainly detected in macrophages in alveoli and in perivascular regions of pulmonary arterioles and venules. Intravenous administration of anti-MCAF/MCP-1 antibodies with MCT significantly decreased macrophage infiltration and eventually reduced the increases in RV/LV+S and RVSP, as well as medial thickening of pulmonary arterioles. Thus, MCAF/MCP-1 is essentially involved in MCT-induced PH by recruiting and activating macrophages.     

Transforming growth factor-beta and receptor tyrosine kinase-activating growth factors negatively regulate collagen genes in smooth muscle of hypertensive rats

Previous studies have suggested that differences in vascular smooth muscle cell (VSMC) proliferative responses between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats can be attributed to transforming growth factor-beta (TGF-beta) actions. Because vascular collagen content is reported to be lower in SHR than in WKY rats, in this study we investigated in cell culture whether the differences in collagen content might also be attributed to differential actions of TGF-beta on VSMCs from the two strains. Exposure of VSMCs from WKY to the TGF-beta isoforms -beta1, -beta2, or -beta3 induced rapid, transient elevations in mRNAs encoding collagens alpha1(I), alpha2(I), and alpha1(III); maximum increases were apparent by 2 hours and ranged from twofold [collagen alpha1(III)] to ninefold [collagen alpha1(I)]. Thereafter they returned to near basal levels. When VSMCs from SHR were exposed to these TGF-beta isoforms, only reductions in collagen mRNA levels were observed, persisting for 24 hours. Basic fibroblast growth factor and epidermal growth factor, factors known to stimulate production of the TGF-beta1 isoform in VSMCs, also induced a pattern of gene responses similar to those induced by the TGF-beta isoforms in VSMCs from SHR and WKY rats. The simultaneous presence of TGF-beta did not affect the time course or magnitude of the changes in collagens alpha1(I), alpha2(I), or alpha1(III) mRNA levels in SHR or WKY VSMCs. Examination of the induction of c-myc mRNA and immunoreactive oncoprotein content indicated that c-myc is a likely contributor to the downregulation of the collagen gene activity in both SHR and WKY VSMCs despite the differential regulation of its mRNA by TGF-beta1 in the two VSMC lines. Together these data suggest that in VSMCs from SHR, a number of gene responses to TGF-beta, in addition to cell proliferation, appear to be abnormal compared with WKY rats, and the lower than normal collagen levels observed in the vasculature of SHR may be in part due to abnormalities in TGF-beta responsiveness.     

Immature rat ovaries become revascularized rapidly after autotransplantation and show a gonadotropin-dependent increase in angiogenic factor gene expression

When the ovaries of 23-day-old juvenile rats are transplanted to an ectopic site, they recover within 1 week the ability to control gonadotropin secretion via steroid negative feedback. Vascular corrosion casting followed by scanning electron microscopy revealed that the transplanted ovary becomes profusely revascularized within 48 h after transplantation. Vascular ingrowth was accompanied by a 40- to 60-fold increase in expression of the genes encoding two angiogenic factors, vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGF beta 1), as assessed by RNA blot hybridization of the corresponding mRNAs. Although TGF beta 3 mRNA levels also increased, no changes in the levels of mRNAs encoding other putative angiogenic factors, such as TGF alpha, basic fibroblast growth factor, and TGF beta 2, were observed. Hybridization histochemistry demonstrated that in intact ovaries, VEGF mRNA is mainly expressed in granulosa cells of the cumulus oophorus and thecal cells of large antral follicles. Transplantation is followed by an increase in mRNA abundance and a dramatic shift in cellular localization, so that the mRNA becomes predominantly expressed in cells of the outer ovarian cortex. In intact ovaries, low levels of TGF beta 1 mRNA were detected in thecal-interstitial cells; after transplantation, its expression also became more predominant in the ovarian outer cortex, but this change was not as marked as in the case of VEGF. Because ovarian autotransplantation is followed by a rapid increase in serum gonadotropin levels, experiments were conducted to determine the importance of this rise in the activation of VEGF and TGF beta 1 gene expression. After transplantation, some animals were treated with the LHRH antagonist Nal-Glu LHRH (50 micrograms/rat, once a day for 2 days) to prevent the posttransplantation rise in serum gonadotropins. Quantitation of VEGF and TGF beta 1 mRNA by RNase protection assay 48 h later showed that suppression of gonadotropin secretion diminished the increase in both VEGF and TGF beta 1 gene expression. Concomitant treatment with PMSG (8 IU/rat, single injection), which mainly bypasses the suppression of endogenous FSH levels, restored the TGF beta 1 mRNA response, but had no effect on VEGF mRNA. The results suggest that the increase in gonadotropin secretion following ovarian transplantation contributes to revascularization of the graft by up-regulating the gene expression of two major angiogenic factors.     

EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension

BACKGROUND: Nitric oxide (NO) and endothelin (ET) have been implicated in the pathogenesis of pulmonary hypertension (PH). Chronic ETA antagonist therapy reduces PH in monocrotaline (MCT)-treated rats. Interactions between the L-arginine-NO pathway and the ET system have been described. We therefore studied the effect of long-term treatment with an oral ETA antagonist (LU 135252) on NO-related vasodilation in isolated lungs from control rats and rats with MCT-induced PH. METHODS AND RESULTS: Three weeks after MCT injection, PH was associated with an increase in right ventricular pressure (from 27.4 +/- 0.9 to 66.6 +/- 4.1 mm Hg) and a decrease in endothelium-independent vasodilation in response to sodium nitroprusside (10(-10) to 10(-5) mol/L; delta Emax, from 11.1 +/- 0.9 to 2.7 +/- 0.3 mm Hg). Endothelium-dependent vasodilation in response to acetylcholine (10(-9) to 10(-4) mol/L) and the calcium ionophore A23187 (10(-9) to 10(-7) mol/L) remained unaffected. Treatment with LU 135252 did not significantly affect the endothelium-dependent and -independent vasodilations in control rats. However, in MCT-treated rats, LU 135252 therapy significantly reduced right ventricular pressure (39.7 +/- 2.1 mm Hg), potentiated acetylcholine-induced vasodilatation (delta Emax, from 1.6 +/- 0.2 to 3.7 +/- 0.4 mm Hg), and improved the responses to sodium nitroprusside (delta Emax, from 2.7 +/- 0.3 to 5.6 +/- 0.6 mm Hg). LU 135252 did not significantly alter the non-receptor-mediated endothelium-dependent vasodilation to A23187 or pulmonary constitutive NO synthase activity. CONCLUSIONS: MCT PH is associated with a reduced smooth muscle responsiveness to NO but a maintained endothelium-dependent vasodilatory potency. Long-term ETA antagonist therapy not only restores smooth muscle responsiveness to NO but also increases endothelium-dependent dilation in response to acetylcholine. This mechanism may contribute to the therapeutic benefit of ETA antagonists in PH.     

Mice with focal pulmonary fibrosis caused by monocrotaline are insensitive to urethane induction of lung tumorigenesis

To establish the characteristics of an optimized pulmonary fibrosis model, male ICR mice were given 4 weekly sc injections of 150 or 0 mg/kg monocrotaline (MCT) and maintained without further treatment for 33 wk (Experiment 1). The final mortality in the MCT group was 64%. Epithelial cells with large bizarre nuclei and an increased incidence of alveolar/bronchiolar hyperplasias were typically observed. In areas of pulmonary fibrosis, the PCNA labeling index (LI) in the alveolar/airway epithelium was significantly elevated. DNA content analysis demonstrated a larger range (4-8C) for the ploidy pattern of alveolar epithelium with large bizarre nuclei than in the normal epithelium (2C). In Experiment 2, the relationship between pulmonary fibrosis development and lung tumorigenesis was investigated. Mice were given 4 weekly sc injections of 150 and 0 mg/kg MCT, followed by a single i.p. injection of 1,000 or 500 mg/kg urethane (UR) on week 7, then maintained without further treatment for an additional 15 wk. UR following MCT-induced inflammatory changes, fibrosis, and epithelia with large bizarre nuclei but no tumorous lesions, in spite of the fact that treatment with UR alone caused a high incidence of pulmonary tumors. Hyperplasias were seen in all groups, but the multiplicity in the combined groups tended to be decreased by the MCT pretreatment. The present study demonstrated that this new protocol is more suitable than previous one for the experimental production of pulmonary fibrosis. Furthermore, the induction of lung tumors by UR was completely depressed in mice with MCT-induced pulmonary fibrosis, suggesting that alveolar epithelial cells are resistant to this lung carcinogen under these conditions.     

Chronic pulmonary hypertension--the monocrotaline model and involvement of the hemostatic system

Monocrotaline (MCT) is a toxic pyrrolizidine alkaloid of plant origin. Administration of small doses of MCT or its active metabolite, monocrotaline pyrrole (MCTP), to rats causes delayed and progressive lung injury characterized by pulmonary vascular remodeling, pulmonary hypertension, and compensatory right heart hypertrophy. The lesions induced by MCT(P) administration in rats are similar to those observed in certain chronic pulmonary vascular diseases of people. This review begins with a synopsis of the hemostatic system, emphasizing the role of endothelium since endothelial cell dysfunction likely underlies the pathogenesis of MCT(P)-induced pneumotoxicity. MCT toxicology is discussed, focusing on morphologic, pulmonary mechanical, hemodynamic, and biochemical and molecular alterations that occur after toxicant exposure. Fibrin and platelet thrombosis of the pulmonary microvasculature occurs after administration of MCT(P) to rats, and several investigators have hypothesized that thrombi contribute to the lung injury and pulmonary hypertension. The evidence for involvement of the various components of the hemostatic system in MCT(P)-induced vascular injury and remodeling is reviewed. Current evidence is consistent with involvement of platelets and an altered fibrinolytic system, yet much remains to be learned about specific events and signals in the vascular pathogenesis.     

Eglin-c prevents monocrotaline-induced ventilatory dysfunction

The present study was carried out to investigate the relationship between elastase and monocrotaline (MCT)-induced ventilatory dysfunction in rats. To accomplish this, we used an elastase inhibitor eglin-c to suppress the activity of endogenous elastase. Thirty-five young Sprague-Dawley rats were randomly divided into six groups: control, MCT, eglin-c(1), eglin-c(2), eglin-c(1) + MCT, and eglin-c(2) + MCT. Rats in the control group received no treatment. Each MCT rat received a single subcutaneous injection of MCT (60 mg/kg) 1 wk before the functional test. Each eglin-c(1) rat was intratracheally instilled with eglin-c (9 mg/rat) twice in 1 wk. Each eglin-c(2) rat was intratracheally instilled with eglin-c (9 mg/rat) five times in 1 wk. Both eglin-c + MCT groups were treated with the combination of eglin-c(1) or eglin-c(2) and MCT. In the MCT group, there were significant decreases in dynamic respiratory compliance, maximal expiratory flow rate at 50% total lung capacity, and the slopes of the maximal expiratory flow-%total lung capacity curve and the maximal expiratory flow-static recoil pressure curve. However, in the eglin-c(1) + MCT and eglin-c(2) + MCT groups, all of the above-mentioned MCT-induced changes were prevented. All ventilatory values of the eglin-c(1) and eglin-c(2) groups were not significantly different from those of the control group. These results demonstrate that eglin-c treatment prevents MCT-induced ventilatory dysfunction and suggest that endogenous elastase may play an important role in MCT-induced inflammation-mediated ventilatory abnormality.     

Increased expression of platelet-derived growth factor receptor alpha and beta and vascular endothelial growth factor in the skin of patients with chronic venous insufficiency

Growth factors produced by a variety of cells act as signalling peptides through specific cell surface receptor pathways. Functions such as cell proliferation, migration and differentiation have been assigned to each of them. Here, we report alterations of platelet-derived growth factor receptor alpha (PDGFR-alpha) and beta (PDGFR-beta) and vascular endothelial growth factor (VEGF) expression patterns in the progressive clinical stages of chronic venous insufficiency (CVI). A total of 30 punch biopsies were taken from patients with CVI, and VEGF and PDGFR were detected by indirect immunofluorescence and immunoperoxidase techniques. PDGFR-alpha and PDGFR-beta expression was strongly increased in endothelial cells of capillaries, pericapillary cells and connective tissue cells in the stroma of the skin of venous eczema and venous leg ulcer patients, and to a smaller extend in the dermis of those with lipodermatosclerosis. VEGF staining showed a similar expression pattern in the progressive CVI stages. However, staining of vessels in particular might simply reflect binding of VEGF, secreted by keratinocytes or fibroblasts, to its receptors. Growth factor and receptor expression in specimens from telangiectases and reticular veins, and from pigmented areas, resembled that of normal skin. We conclude that PDGFR-alpha, PDGFR-beta and VEGF play an important role in mediating inflammation and epithelial hyperproliferation in venous eczema, inducing connective tissue sclerosis in lipodermatosclerosis, and causing the reduced reepithelialization tendency in venous ulcers. We speculate that endothelial proliferation with chronic venous hypertension might be mediated by these growth factors.     

Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension

Vascular endothelial growth factor (VEGF) is a potent mitogenic and permeability factor targeting predominantly endothelial cells. At least two tyrosine kinase receptors, Flk-1 and Flt-1, mediate its action and are mostly expressed by endothelial cells. VEGF and VEGF receptor expression are upregulated by hypoxia in vivo and the role of VEGF in hypoxia-induced angiogenesis has been extensively studied in a variety of disease entities. Although VEGF and its receptors are abundantly expressed in the lung, their role in hypoxic pulmonary hypertension and the accompanying vascular remodeling are incompletely understood. We report in this in vivo study that hypoxia increases mRNA levels for both VEGF and Flk-1 in the rat lung. The kinetics of the hypoxic response differ between receptor and ligand: Flk-1 mRNA showed a biphasic response to hypoxia with a significant, but transient, rise in mRNA levels observed after 9-15 h of hypoxic exposure and the highest levels noted after 3 wk. In contrast, VEGF mRNA levels did not show a significant increase with acute hypoxia, but increased progressively after 1-3 wk of hypoxia. By in situ hybridization, VEGF mRNA was localized predominantly in alveolar epithelial cells with increased signal in the lungs of hypoxic animals compared with controls. Immunohistochemical staining with anti-VEGF antibodies localized VEGF peptide throughout the lung parenchyma and was increased in hypoxic compared with normoxic animals. Furthermore, hypoxic animals had significantly higher circulating VEGF concentrations compared with normoxic controls. Lung vascular permeability as measured by extravasation of Evans Blue dye was not significantly different between normoxic and hypoxic animals, although a tendency for increased permeability was seen in the hypoxic animals. These findings suggest a possible role for VEGF in the pulmonary response to hypoxia.